The invention generally relates to mass spectrometers and specifically to tandem mass spectrometers. More specifically the invention is directed to a mass spectrometry apparatus and method that provides an effective solution for multiple stage mass spectrometric analysis and coupling of low resolution multiple stage mass spectrometry devices with external high-resolution mass spectrometers.
Traditionally tandem mass spectrometers (MS-MS) have been employed to provide structural information for samples of interest. In MS-MS instruments, a first mass spectrometer is used to select a primary ion of interest, for example, a molecular ion of a particular biomolecular compound such as a peptide, and that ion is caused to fragment by increasing its internal energy, for example, by colliding the ion with a neutral molecule. A second mass spectrometer then analyzes the spectrum of fragment ions, and often the structure of the primary ion can be determined by interpreting the fragmentation pattern. The MS-MS instrument improves the recognition of a compound with a known pattern of fragmentation and also improves specificity of detection in complex mixtures, where different components give overlapping peaks in simple MS. In the majority of applications the detection limit is defined by the level of chemical noise. Drug metabolism studies and protein recognition in proteome studies are good examples. Frequently, MS-MS techniques can also improve the detection limit. When analyzing certain samples it is often desirable to conduct further analyses of fragments produced from the originally selected ion, and such further analyses consist of repeated sequences of mass to charge ratio (m/z) isolation and fragmentation. In some cases different m/z fragments derived from a single parent ion are further analyzed, and in other cases a single m/z fragment is subjected to a succession of xe2x80x9cnxe2x80x9d mass spectrometric steps to yield relevant information regarding the original parent ion. This continued mass spectrometric analyses is referred to in the art as MSn analyses. Various types of mass spectrometers have been employed to conduct MSn analysis as discussed below.
A three-dimensional ion trap (3-D IT) is one of the most flexible devices for MS-MS and multi-step (MSn) analysis. This trap is composed of a ring electrode and two end cap electrodes of special shape to create a quadrupolar distribution of potential. Radio frequency (RF) and DC offset electric potentials are applied between electrodes and cause ions to oscillate within the trap. By appropriately selecting voltage parameters, ions of a specific mass/charge ratio can be made to have stable or unstable trajectories. In another implementation an additional (auxiliary) AC voltage is applied to the end-caps to induce resonant excitation of selected ions either for the purpose of ejecting the selected ions or for the purpose of inducing collisional dissociation.
The 3-D ion trap is capable of single step mass spectrometric analysis. In such analysis ions are injected into the trap (or generated within the trap), confined to the center of trap because of low energy collisions with an inert gas such as helium (typically at 1 mtorr pressure) and then sequentially ejected through the apertures in the end cap electrodes onto an external detector by raising the amplitude of the RF field. The same device could be used for a multi-step, i.e. MSn , analysis. The ion trap isolates ions in a m/z window by rejecting other components, then fragments these isolated ions by AC excitation, then isolates resulting ion fragments in a m/z window and repeats such sequence (MSn operation) in a single cell. At the end of the sequence ions are resonantly ejected to acquire the mass spectrum of N-th generation fragments. The 3-D IT is vulnerable to sensitivity losses due to ion rejection and instability losses at the time of ion selection and fragmentation.
Fourier transform ion cyclotron resonance mass spectrometry (FTMS) currently provides the most accurate measurement of ion mass to charge ratios with a demonstrated resolution in excess of 100,000. In FTMS, ions are either injected from outside the cell or created inside the cell and confined in the cell by a combination of static magnetic and electric fields (Penning trap). The static magnetic and electric field define the mass dependent frequency of cyclotron motion. This motion is excited by an oscillating electric potential. After a short period the applied field is turned off. Amplifying and recording weak voltages induced on the cell plates by the ion""s motion detects the frequency of ion motion and, thus, the m/z of the ion. Ions are selectively isolated or dissociated by varying the magnitude and frequency of the applied transverse RF electric potential and the background neutral gas pressure. Repeated sequences of ion isolation and fragmentation (MSn operation) can be performed in a single cell. An FIMS is a xe2x80x9cbulkyxe2x80x9d device occupying a large footprint and is also expensive due to the costs of the magnetic field. Moreover, an FTMS exhibits poor ion retention in MSn operation (relative to the 3-D ion trap).
Currently, the most common form of tandem mass spectrometer is a triple quadrupole, where both mass spectrometers are quadrupoles and an RF only quadrupole functions as a collisional cell to enhance ion transport. Because of low scanning speed the instrument employs continuous ion sources like ESI and atmospheric pressure chemical ionization (APCI). Since scanning the second mass spectrometer would cause losses, the most effective way of using this instrument is monitoring of selected reactions. Drug metabolism studies are a good example where a known drug compound is measured in a rich biological matrix, like blood or urine. In those studies both parent and daughter fragment masses are known and the spectrometer is tuned on those specific masses. For more generic applications requiring scanning, the triple quadrupole instrument is a poor instrument choice because of its low speed, sensitivity, mass accuracy and resolution.
Recently hybrid instruments combining quadrupoles with time of flight analyzers (Q-TOF) have been described where the second quadrupole mass spectrometer is replaced by an orthogonal time of flight spectrometer (o-TOF). The o-TOF back end allows observation of all fragment ions at once and the acquisition of secondary spectra at high resolution and mass accuracy. In cases where the full mass range of daughter ions is required, for example, for peptide sequencing, the Q-TOF strongly surpasses the performance of the triple quadrupole. However, the Q-TOF suffers a 10 to a 100 fold loss in sensitivity as compared to a single quadrupole mass filter operating in selected reaction monitoring mode (monitoring single m/z). For the same reason the sensitivity of the Q-TOF is lower in the mode of xe2x80x9cparent scanxe2x80x9d where, again, the second MS is used to monitor a single m/z.
More recently, the quadrupole has been replaced by a linear ion trap (LIT). The quadrupole with electrostatic xe2x80x9cplugsxe2x80x9d is capable of trapping ions for long periods of time. The quadrupole field structure allows one to apply an arsenal of separation and excitation methods, developed in 3-D ion trap technology, combined with easy introduction and ejection of the ion beam out of the LIT. The LIT eliminates ion losses at selection and also can operate at poor vacuum conditions which reduces requirements on the pumping system. However, a limited resolution of ion selection, R less than 200, has been demonstrated thus far.
All of the existing MSn devices suffer from a common drawback in that they do not provide a capability for storing results of multiple MS steps with the concomitant capability to explore multiple branches of fragmentation using the same ion material. The current state of the art is that the sequence of functional steps (selection, cooling, fragmentation and analysis) is done either xe2x80x9cin-timexe2x80x9d while keeping results in the same cell, as in ion trap and FTMS devices, or xe2x80x9cin-spacexe2x80x9d with the ion beam constantly flowing, as in triple quadrupole, Q-TOF and LIT-TOF devices. In both cases selection of ions of interest automatically means rejection of all other components. As a result the existing instruments limit the number of multi-step MS analysis that can be carried out simply due to ion losses. It would therefore be desirable to improve the sensitivity of MSn analysis and thus provide a capability of detailed sequencing and analysis of ultra low quantity sample in complex matrixes. It would also be desirable to produce a device with multiple cells for storing the results of each step of the MSn analysis and thereby provide a device with a high efficiency of selection and fragmentation.
The present invention overcomes the disadvantages and limitations of the prior art by providing a highly sensitive multiple stage (MSn ) mass spectrometer and mass spectrometric method, capable of eliminating losses of ions during the isolation stage. Ions of interest are physically isolated (by m/z value) without rejecting ions of other m/z values, so that the selected ions may be dissociated, while the rest of the ion population is available for subsequent isolation, dissociation and mass spectrometric analysis of fragment ions.
A preferred embodiment of the invention includes a pulsed ion source coupled with a linear array of mass selective ion trap devices at least one of the traps being coupled to an external ion detector. Each ion trap device is configured with a storing cell for ion trapping interspersed between a pair of guarding cells, all aligned along a common axis, denoted in the following as the z direction (FIG. 1). A combination of radio frequency (RF) and direct current (DC) voltages are applied to electrodes of the ion trap device to retain ions within the trapping (storing) cells. Each trapping cell has a sub-region in which the dynamical motion of the ion exhibits resonance frequencies along the z direction. These resonance frequencies are m/z-dependent so that the ion motion can be selectively excited by m/z value through the application of AC voltages to various electrodes of the ion trap device. The AC voltages can be combined with time-resolved changes in the applied DC voltages so that each individual trapping cell can be switched between ion trapping, mass selecting and ion fragmenting modes. Ions may be selectively transferred between traps of said linear array, and selectively dissociated within each trap of said linear array to enable a higher sensitivity MSn operation. The application of the RF, AC and DC voltages and the resulting modes of operation of the invention depend on specific embodiments of the general concept as will be described in detail below.
The present invention is applicable to all currently useful methods of ion generation. In one preferred embodiment the pulsed ion source comprises an intrinsically pulsed (MALDI) ion source. In another preferred embodiment the pulsed ion source comprises an electrospray (ESI) or an atmospheric pressure chemical ionization (APCI) ion source with a storing multipole guide (for example, an accumulating quadrupole), periodically injecting ions into the array of ion traps.
In accordance with embodiments of the invention, the final mass analysis of fragments of the nth generation of fragmentation can be done either by mass dependent ejection of ions from the last (i.e., furthest from the ion source) ion trap within the array of ion trap devices onto a detector or by introducing the entire ion content of any cell into an external mass spectrometer of conventional design. In one particular embodiment, the external mass spectrometer is a time-of-flight mass spectrometer (TOF MS). In a preferred case of this embodiment, ions are pulsed injected into an orthogonal TOF MS with a synchronized orthogonal pulsing to reduce so-called xe2x80x9cduty-cyclexe2x80x9d losses. In yet another particular case of the embodiment, the last cell of the linear ion trap serves as an acceleration stage for the TOF MS.
In yet another embodiment, the mass spectrum of ions and/or fragments is acquired by measuring the weak electric signal induced by ion oscillations on the confining electrodes that are part of the ion trap cells.
In accordance with the invention, each ion trapping device with the linear array of ion trapping devices can be generally classified by the nature of the linear approximation to the ion trapping field in the center of the device (the xe2x80x98originxe2x80x99). An ion trapping field of a certain linear approximation can be realized by multiple electrode geometries and applied AC and DC signals. In one preferred embodiment, the linear approximation to the ion trapping field generates a harmonic linear trap (HLT) device. In another preferred embodiment, the linear approximation to the ion trapping field generates a Paul trap device.
The origin of the HLT or Paul trap device is the point inside the trapping region where the electric field for trapping a single ion vanishes. In the vicinity of the origin for the HLT or Paul trap the equations of motion for a single ion can be approximated by a linear set of three 2nd order, ordinary differential equations of motion. The HLT class covers three-dimensional ion traps in which a harmonic oscillator equation governs one coordinate (by convention, the z coordinate) and Mathieu equations govern the x and y coordinates. The Paul trap class has Mathieu equations governing all three coordinates.
In a preferred embodiment, the HLT device is configured as a triplet of electrode cells. Each cell triplet consists of open parallelpiped cells surrounding an open parallelpiped trapping cell. In the linear array, a guarding cell is shared between adjacent HLT triplets. The guarding and storing cells are distinguished by length, DC offset, z excitation voltages (dipolar) and function. In this embodiment the gate and trapping cells share the radial trapping RF voltage.
In yet another preferred embodiment, for the purpose of achieving a higher resolution of mass selection, each cell of the linear array is a Paul trap. The basic electrode geometry for the Paul trap could be the same as the HLT discussed above or can be constructed using hollow cylinder cells interspersed between guarding plate electrodes. In each instance, unlike the HLT, the RF is applied only to the trapping (storing) cells. A DC voltage is applied to both trapping and guarding cells and an AC signal (for exciting the z motion of the ions) is applied between the guarding and trapping cells.
A method of multiple step mass spectrometric analysis according to the present invention includes pulsed introduction of an ion beam into one of a plurality of multiple communicating ion traps, combined with novel features including a) sampling of ions into the adjacent trap without losses of other components, b) storing ion fragments of each generation in individual traps, and c) using stored ions for subsequent analysis of multiple fragmentation channels. Sampling a portion of the ions into an external mass spectrometer allows the extensive use of economic data-dependent algorithms in the selective transfer and dissociation of ions in the device.
In accordance with a preferred embodiment, the method of selective ion transfer between trapping cells involves an array of either HLT or Paul trap devices and further involves applying an AC signal between the guarding and storing cells (dipolar field local to the origin) with a single frequency equal to the resonant frequency of the ion""s z motion. In one particular case of the embodiment, the applied AC signal has a time-dependent frequency that tracks the frequency shift in the ion""s resonant z motion due to nonlinear electric fields perturbing the ion""s motion away from the origin.
In an array of HLT the selective transfer method can be improved by lowering the DC barrier between ion traps once the ions of a predetermined m/z value are AC excited to the highest energy within the ion population in the trap. It is advantageous to drop the DC barrier at a predetermined phase of ion oscillation.
One aspect of the above-described methods of selective ion transfer is that non-transferred ions are retained within the ion trap for subsequent analysis. This is in contrast to conventional selection methods, where ions are isolated by the ejection of other components.
A method of ion fragmentation in accordance with one embodiment of the present invention is accomplished by accelerating ions between cells either by using a DC electric field between cells or by resonant AC excitation of ions. Contrary to fragmentation within a Paul trap, the ion fragmentation in the HLT array is characterized by minimal ion losses due to the greater stability of ion motion in the radial direction.